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J Mol Biol. 1993 Oct 5;233(3):536-45.

A novel allosteric mechanism in haemoglobin. Structure of bovine deoxyhaemoglobin, absence of specific chloride-binding sites and origin of the chloride-linked Bohr effect in bovine and human haemoglobin.

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1
M.R.C. Laboratory of Molecular Biology, Cambridge, England.

Abstract

The structure of bovine deoxyhaemoglobin has been determined at 2.2 A resolution and refined to an R-factor of 0.193 for all 32,583 reflections, and a free R-factor of 0.249 for 1527 reflections excluded from the refinement. The structure shows no significant differences between the alpha-carbon positions of bovine and human haemoglobin, except at the N-terminal segment and the first helix (A) which are closer to the dyad symmetry axis and pushed more tightly against the rest of the beta-subunits in the bovine form. In a search for the predicted chloride-binding sites, three-dimensional data were collected from crystals suspended in 50% polyethylene glycol buffered either with 50 mM Na phosphate (pH 7.3) +/- 0.1 M NaCl or with 0.1 M Hepes (pH 7.3) +/- 0.1 M NaBr. Difference electron density maps with and without NaCl or NaBr showed no evidence of specific halide ion-binding sites. Oxygen equilibria were measured in 10 mM Hepes buffer without added NaCl, with 0.1 mM NaCl, 0.1 M NaCl + 1 mM 2,3-diphosphoglycerate, and 0.1 M NaCl + 1 mM inositol hexaphosphate. Without added chloride, P50 of stripped bovine haemoglobin was similar to that of human haemoglobin with 0.1 M NaCl. With 0.1 M NaCl it was similar to that of human haemoglobin saturated with 2,3-diphosphoglycerate. In 0.1 M NaCl neither organic phosphate significantly affected the oxygen affinity. Titration of P50 with NaCl showed delta log P50/delta log[Cl-] of bovine and human haemoglobin to be identical. Analysis of the oxygen equilibrium curves showed the low intrinsic oxygen affinity of bovine haemoglobin to be due to a larger oxygen dissociation constant from the T-structure. The influence of chloride on P50 and on the alkaline Bohr effect is the same in bovine and human haemoglobins. It is proposed that this is due to the excess positive charges in the central cavity and its widening in the transition from the R to the T-structure. The widening would allow more chloride ions to enter and neutralize the positive charges, but these ions would remain mobile and therefore do not show up as peaks of high electron density. Repulsion between excess positive charges in the central cavity raises the free energy of the T-structure relative to the R-structure, thereby raising the oxygen affinity. Conversely, entry of chloride ions on widening of the cavity reduces the free energy of the T-structure and therefore lowers the oxygen affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID:
8411160
DOI:
10.1006/jmbi.1993.1530
[Indexed for MEDLINE]
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